U.S. patent application number 10/466094 was filed with the patent office on 2004-05-20 for selectively hybridizable substance immobilization fiber, fiber array comprising bundle of such fibers, selective hybridizing method, device therefor, and base.
Invention is credited to Higasa, Masashi, Nagino, Kunihisa, Nobumasa, Hitoshi, Sone, Saburo, Watanabe, Koji.
Application Number | 20040096169 10/466094 |
Document ID | / |
Family ID | 18873772 |
Filed Date | 2004-05-20 |
United States Patent
Application |
20040096169 |
Kind Code |
A1 |
Sone, Saburo ; et
al. |
May 20, 2004 |
Selectively hybridizable substance immobilization fiber, fiber
array comprising bundle of such fibers, selective hybridizing
method, device therefor, and base
Abstract
Disclosed is an array immobilizing selective binding substances
thereon, with which each sample can be distinguished without
relying on the positions, an arbitrary array may be made, the
density may be freely changed depending on the method of using, and
with which the reaction between the bound sample and another sample
can be detected easily. The present invention provides a fiber on
which a selective-binding substance is immobilized, or a fiber
array comprising a bundle of the fibers. A method for binding
reaction between a selective binding substance and a corresponding
selective binding substance, in which a test sample and/or the
corresponding selective binding substance is(are) moved relative to
the surface on which the selective binding substance is immobilized
by, for example, applying an AC voltage in a direction crossing the
perpendicular axis of the surface on which the selective binding
substance is immobilized was also provided.
Inventors: |
Sone, Saburo; (Yokohama-shi,
JP) ; Nagino, Kunihisa; (Moriyama-shi, JP) ;
Higasa, Masashi; (Takatsuki-shi, JP) ; Nobumasa,
Hitoshi; (Otsu-shi, JP) ; Watanabe, Koji;
(Kusatsu-shi, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
18873772 |
Appl. No.: |
10/466094 |
Filed: |
December 11, 2003 |
PCT Filed: |
January 11, 2002 |
PCT NO: |
PCT/JP02/00115 |
Current U.S.
Class: |
385/115 |
Current CPC
Class: |
B01J 19/0046 20130101;
B01J 2219/00605 20130101; B01J 2219/00612 20130101; C40B 40/10
20130101; B01J 2219/00664 20130101; B01J 2219/0074 20130101; B01J
2219/00596 20130101; G01N 33/54366 20130101; B01J 2219/0061
20130101; B01J 2219/00673 20130101; B01J 2219/00497 20130101; B01J
2219/00585 20130101; B01J 2219/00637 20130101; B01J 2219/00722
20130101; B01J 2219/00725 20130101; B01J 2219/00515 20130101; B01J
2219/00626 20130101; B01J 2219/00729 20130101; C40B 40/06 20130101;
B01J 2219/00524 20130101; G01N 33/54313 20130101; B01J 2219/00659
20130101; B01J 2219/00576 20130101; B01J 2219/00621 20130101; B01J
2219/00677 20130101 |
Class at
Publication: |
385/115 |
International
Class: |
G02B 006/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 12, 2001 |
JP |
2001-005775 |
Claims
1. A fiber on which a selective-binding substance is immobilized,
or a fiber array comprising a bundle of said fibers.
2. The fiber or the array comprising a bundle of said fibers
according to claim 1, wherein said selective-binding substance is a
nucleic acid, protein, saccharide, or an antigenic compound.
3. The fiber or the array comprising a bundle of said fibers
according to claim 1, wherein said selective-binding substance is a
nucleic acid, antibody or an antigen.
4. The fiber or the array comprising a bundle of said fibers
according to claim 1, wherein said fiber transmits light.
5. The fiber and the array comprising a bundle of said fibers
according to claim 1, wherein said fiber is electrically
conductive.
6. The fiber and the array comprising a bundle of said fibers
according to claim 1, wherein said fiber is flexible.
7. The fiber and the array comprising a bundle of said fibers
according to claim 1, wherein said selective-binding substance is
immobilized on an end of said fiber.
8. The fiber array according to claims 1 to 7, wherein all of said
fibers or a part of said fibers in said fiber array carry different
types of said selective binding substance.
9. The fiber array according to claim 8, wherein said fibers on
which said selective binding substances are immobilized can be
distinguished from each other.
10. The fiber array according to any one of claims 1 to 9, wherein
a substance reacted with said immobilized selective binding
substance can be directly detected through said fiber.
11. The fiber array according to any one of claims 1 to 9, wherein
said fiber is an optical fiber.
12. A method for measuring the result of a selective binding
reaction, comprising treating said fiber array according to any one
of claims 1 to 11 with a test sample containing a corresponding
selective binding substance which selectively binds to said
selective binding substance immobilized on said fiber in said fiber
array, and measuring said corresponding selective binding substance
bound to said fiber after washing the fibers.
13. The method according to claim 12, wherein said fiber transmits
light, and said selective binding substance is immobilized on an
end of said fiber.
14. The method according to claim 13, wherein said corresponding
selective binding substance is labeled with a fluorescent or
luminescent substance, or is one which selectively binds to a
substance for measurement labeled with a fluorescent or luminescent
substance, and after reaction and washing, measuring the result of
the reaction by measuring the fluorescence or luminescence from
said label attached to said corresponding selective binding
substance or said substance for measurement from another end of
said fiber.
15. A method for selectively binding said selective binding
substance immobilized on said fiber in said fiber array according
to claim 5 with a test sample containing a corresponding selective
binding substance, comprising applying electric voltage or electric
current to said fiber array according to claim 5.
16. A method for binding reaction between a selective binding
substance and a corresponding selective binding substance,
comprising immobilizing said selective binding substance on a
substrate; and making a test sample contacted to said immobilized
selective binding substance, which test sample contains said
corresponding selective binding substance which selectively binds
to said selective binding substance, thereby carrying out said
binding reaction; said test sample and/or said corresponding
selective binding substance being moved relative to the surface on
which said selective binding substance is immobilized, in said step
of immobilizing said selective binding substance on a substrate and
making a test sample contacted to said immobilized selective
binding substance.
17. The method according to claim 16, wherein said binding reaction
is carried out while applying an AC voltage in a direction crossing
the perpendicular axis of the surface on which said selective
binding substance is immobilized, between electrodes arranged
outside the both ends of the area on which said selective binding
substance is immobilized, in said step of immobilizing said
selective binding substance on a substrate and making a test sample
contacted to said immobilized selective binding substance, which
test sample contains said corresponding selective binding substance
which selectively binds to said selective binding substance,
thereby carrying out said binding reaction.
18. The method according to claim 16 or 17, wherein a plurality of
areas each on which said selective binding substance is immobilized
exist, and a selective binding substance-arrayed region in which
said areas are arranged exists, and said electrodes are arranged
outside said selective binding substance-arrayed region.
19. The method according to any one of claims 16 to 18, wherein
said selective binding substance is at least one selected from the
group consisting of nucleic acids, proteins, saccharides,
antibodies and antigenic compounds.
20. The method according to claim 19, wherein said selective
binding substance and said corresponding selective binding
substance are single-stranded nucleic acids, and said binding
reaction is hybridization between the nucleic acids.
21. An apparatus for conducting a binding reaction including a step
of immobilizing a selective binding substance on a substrate and
making a test sample contacted to said immobilized selective
binding substance, which test sample contains a corresponding
selective binding substance which selectively binds to said
selective binding substance, thereby carrying out said binding
reaction, said apparatus comprising a base on which said substrate
is mounted; electrodes arranged in a direction crossing the
perpendicular axis of the surface on which the selective binding
substance is immobilized, and arranged outside the both ends of the
area on which said selective binding substance is immobilized; and
means for applying AC voltage between said electrodes.
22. A substrate for conducting a binding reaction including a step
of immobilizing said selective binding substance on said substrate
and making a test sample contacted to said immobilized selective
binding substance, which test sample contains said corresponding
selective binding substance which selectively binds to said
selective binding substance, thereby carrying out said binding
reaction between said selective binding substance and said
corresponding selective binding substance, said substrate
comprising a site for immobilizing said selective binding
substance, on which said selective binding substance on said
substrate is immobilized; and electrodes arranged outside the both
ends of region for immobilizing said selective binding
substance.
23. The substrate according to claim 22, wherein a plurality of
sites for immobilizing said selective binding substance exist, and
a selective binding substance-arrayed region in which said sites
are arranged exists, and said electrodes are arranged outside said
selective binding substance-arrayed region.
24. The substrate for immobilizing the selective binding substance
according to claim 22 or 23, characterized in that said site for
immobilizing said selective binding substance is a protruded or
hollow site.
25. The substrate according to claim 24, wherein said site for
immobilizing said selective binding substance is an end of a fiber
or a bundle of fibers, which fiber or bundle is inserted through a
hole formed in said substrate.
26. The substrate according to any one of claims 22 to 25, wherein
said selective binding substance is immobilized on said site for
immobilizing said selective binding substance.
27. The substrate according to any one of claims 22 to 26, wherein
said selective binding substance is at least one selected from the
group consisting of nucleic acids, proteins, saccharides,
antibodies and antigenic compounds.
28. The substrate according to claim 27, wherein said selective
binding substance and said corresponding selective binding
substance are single-stranded nucleic acids, and said binding
reaction is hybridization between the nucleic acids.
29. The substrate according to any one of claims 22 to 28, wherein
said electrodes are made of at least one material selected from the
group consisting of simple metals selected from platinum, gold,
silver, aluminum, copper and palladium, alloys thereof, carbon and
carbon compounds, and electrically conductive polymers.
Description
TECHNICAL FIELD
[0001] The present invention relates to a fiber on which a
substance that selectively binds to a test substance (referred to
as "selective binding substance" in the present specification), and
a fiber array containing a bundle of the fibers, and method for
selective binding reaction between the selective binding substance
and a corresponding selective binding substance which selectively
binds thereto, and apparatus and substrate therefor.
BACKGROUND ART
[0002] Studies of analysis of genetic information of various
organisms have been started, and a number of genes including human
genes and nucleotide sequences thereof, and information about
proteins encoded by the genes and sugar chains secondarily prepared
from the proteins are now rapidly disclosed. Functions of polymers
such as genes of which sequences have been clarified, proteins and
sugar chains may be investigated by various methods. As for main
methods for nucleic acids, the relationships between various genes
and their expressed functions in the body may be investigated
utilizing complementarity between various nucleic acids/nucleic
acids, by a method such as Northern hybridization or Southern
hybridization. As for proteins, the functions and expression of
proteins may be investigated utilizing protein/protein reactions,
as represented by Western hybridization.
[0003] In recent years, new methods or methodology called DNA
microarray method (DNA chip method) has been developed as methods
for analyzing expression of a number of genes at one time, and the
methods are drawing attention. The principle of these methods are
the same as that of the conventional methods in the respect that
they are methods for detecting and quantifying nucleic acids
utilizing nucleic acid/nucleic acid hybridization. These methods
may also be applied to the detection and quantification of proteins
and sugar chains based on the binding reactions between
protein/protein, between sugar chain/sugar chain and between sugar
chain/protein. These technologies are mainly characterized in that
a number of DNA fragment, proteins or sugar chains are immobilized
in an array on a planar substrate called microarray or chip. As for
the concrete method for using the microarray, for example, genes
expressed in the cells of interest are labeled with a fluorescent
dye to prepare a sample, the sample is applied to the planar
substrate so as to allow binding between complementary nucleic
acids (DNA or RNA), the site is labeled with a fluorescent dye or
the like and then the positions are read at a high speed using a
high resolution analyzer, or the response based on an
electrochemical reaction such as electric current or the like is
detected. By these methods, the types of genes contained in the
sample may be quickly identified.
[0004] As for the technique for immobilizing a nucleic acid on the
substrate, methods in which poly-L-lysine, aminosilane or the like
is coated on the substrate made of a glass or the like, and the
nucleic acids are immobilized thereon, and the like have been
developed to further increase the density, in addition to the
methods in which the nucleic acids are immobilized at a high
density on a Nylon sheet or the like in the same manner as in the
above-mentioned Northern method.
[0005] However, it is pointed out that by the methods in which
nucleic acids are spotted on a substrate prepared by chemically or
physically modifying a solid surface such as glass, the density of
the spots and the amount of the nucleic acid which can be
immobilized per one spot are small, and the reproducibility is
poor. Further, it is said that a large cost down per one chip is
difficult due to an expensive production apparatus and production
process including a number of steps. Further, since the
distinguishment between the nucleic acids must be relied on the
positions of the spots, so that the arrangement and distinguishment
of the spots on the substrate are not simple.
[0006] As for the microarray using proteins and sugar chains, the
same problems as those using nucleic acids exist.
[0007] As a method which does not utilize the planar substrate,
methods utilizing microbeads are known. However, these methods are
incomplete in the distinguishment of the various types of
microbeads each on which the respective nucleic acid, protein or
sugar chain is immobilized, and it is difficult to prepare one in
which the designated compounds are arrayed with a designated
sequence with good reproducibility.
[0008] On the other hand, it has been tried to immobilize nucleic
acids in hollow fibers (EP-A-1162262). According to this technique,
by immobilizing a nucleic acid in one hollow fiber so as to make a
relationship such that one nucleic acid corresponds to one fiber,
each of the nucleic acids is distinguished. However, with this
technique, the hollow fibers are finally bundled, and each nucleic
acid is recognized based on the positional relationship of the
fibers, so that the problem in the method in which the nucleic
acids are spotted on a substrate is not overcome.
[0009] Since the nucleic acid solutions used for nucleic
acid/nucleic acid hybridization are valuable, it is desired to
carry out the hybridization reaction using the nucleic acids in
amounts as small as possible. To this end, it is thought to
decrease the concentrations of the nucleic acids in the nucleic
acid solutions. To attain a good efficiency of hybridization even
when the concentrations of the nucleic acid solutions are small, it
is tried to promote the efficiency of hybridization by arranging
the specimen nucleic acids-immobilized sites on a substrate of the
above-mentioned microarray, which substrate has an electrically
conductive layer; generating electric field by applying positive
potential to the specimen nucleic acids-immobilized sites, thereby
attracting the sample nucleic acids in the nucleic acid solution to
the vicinity of the specimen nucleic acid-immobilized sites so as
to increase the local nucleic acid concentration in the vicinity of
the specimen nucleic acid-immobilized sites (Japanese Laid-open
Patent Application (Kokai) No. 8-154656).
[0010] As for microarrays using proteins or sugar chains, the
effect similar to the microarrays using nucleic acids is
expected.
[0011] However, the effect to promote measurement sensitivity or to
shorten the hybridization time are not satisfactory, so that a more
effective hybridization method is demanded.
DISCLOSURE OF THE INVENTION
[0012] Under these circumstances, establishment of a new systematic
methodology by which macromolecules may be immobilized to a
prescribed concentration and may be arrayed in a measurable manner
at a high density with a good reproducibility, and by which each of
the macromolecules may be recognized without relying on the
positions thereof, and which may be applied to inexpensive mass
production is strongly demanded for the analysis of macromolecules,
that is thought to increase its importance. This is an object of
the present invention.
[0013] More particularly, an object of the present invention is to
provide means by which each sample may be distinguished without
relying on the positions, an arbitrary array may be made, the
density may be freely changed depending on the method of using, and
by which the reaction between the bound sample and another sample
can be detected easily, when compared with the process for
producing macromolecule array by microspotting or micro-pouring on
a two-dimensional carrier such as nylon sheet or glass.
[0014] Further, establishment of a method for selective binding
reaction, by which valuable macromolecule samples such as nucleic
acids, proteins, sugar chains, antibodies and antigens may be
utilized in small amounts and effectively is strongly demanded for
the analysis of macromolecules, that is thought to increase its
importance. This is also an object of the present invention.
[0015] More particularly, with the conventional microarrays such as
those in which a number of selective binding substances such as DNA
fragments, proteins or sugar chains are immobilized in an array at
a high density; those prepared by bundling the porous hollow fibers
in which the selective binding substances are immobilized, cutting
the resultant in the direction crossing the fiber axis to prepare
thin pieces to constitute a two-dimensional high density fiber
array in which the selective binding substances are immobilized on
the surfaces of the fibers; or those in which the selective binding
substances are immobilized on the surfaces of arrayed fibers, and
the fibers are arrayed in a three-dimensional structure; the
hybridization reaction is relied on the natural diffusion of the
selective binding substance, so that it is difficult to cause the
hybridization reaction effectively using a solution containing
small amounts of selective binding substances, and to effectively
utilize the valuable selective binding substances. Even with the
method for effectively carrying out the hybridization reaction of
the selective binding substances utilizing electric attraction,
which was invented for overcoming the inefficiency of the
conventional hybridization methods, the promotion of efficiency is
not sufficient.
[0016] In view of this, an object of the present invention is to
provide a method for selective binding reaction, which overcomes
the above-mentioned drawbacks of the prior art and which causes the
selective binding reaction effectively utilizing small amounts of
selective binding substances, and to provide an apparatus and
substrate therefor.
[0017] The present inventors intensively studied to abolish the
conventional concept by which the arraying process of the selective
binding substances and the immobilization process are conducted on
the two-dimensional carrier, and to discover that a fiber bundle
may be provided, with which each fiber therein on which each sample
is immobilized is distinguished without relaying on its position in
a three-dimensional structure, by conducting the immobilization
process independently on a one-dimensional fiber (on one fiber)
which can be distinguished based on the support, magnetism, bar
code, color or shape.
[0018] Further, the present inventors discovered that a fiber or
array of fibers with which the substance reacted with the selective
binding substance immobilized on the fiber can be directly detected
may be prepared, by using a light-transmitting material or
electrically conductive material for constituting the fibers,
thereby completing the present invention.
[0019] That is, the present invention provides a fiber on which a
selective-binding substance is immobilized, or a fiber array
comprising a bundle of the fibers.
[0020] The present inventors further intensively studied for
attaining the above-mentioned object to discover that the
efficiency of the hybridization reaction may be promoted by
increasing the probability of collision between the selective
binding substance and the corresponding selective binding substance
by moving the corresponding selective binding substance in the
vicinity of the selective binding substance immobilized on the
microarray substrate or on the fiber, thereby completing the
present invention.
[0021] That is, the present invention provides a method for binding
reaction between a selective binding substance and a corresponding
selective binding substance, comprising immobilizing the selective
binding substance on a substrate; and making a test sample
contacted to the immobilized selective binding substance, which
test sample contains the corresponding selective binding substance
which selectively binds to the selective binding substance, thereby
carrying out the binding reaction; the test sample and/or the
corresponding selective binding substance being moved relative to
the surface on which the selective binding substance is
immobilized, in the step of immobilizing the selective binding
substance on a substrate and making a test sample contacted to the
immobilized selective binding substance. The present invention also
provides an apparatus for conducting a binding reaction including a
step of immobilizing the selective binding substance on a substrate
and making a test sample contacted to the immobilized selective
binding substance, which test sample contains the corresponding
selective binding substance which selectively binds to the
selective binding substance, thereby carrying out the binding
reaction, the apparatus comprising a base on which the substrate is
mounted; electrodes arranged in a direction crossing the
perpendicular axis of the surface on which the selective binding
substance is immobilized, and arranged outside the both ends of the
area on which said selective binding substance is immobilized; and
means for applying AC voltage between the electrodes. The present
invention still further provides a substrate for conducting a
binding reaction including a step of immobilizing the selective
binding substance on the substrate and making a test sample
contacted to the immobilized selective binding substance, which
test sample contains the corresponding selective binding substance
which selectively binds to the selective binding substance, thereby
carrying out the binding reaction, the substrate comprising a site
for immobilizing the selective binding substance; and electrodes
arranged outside the both ends of region for immobilizing the
selective binding substance.
[0022] By the present invention, a fiber on which a
selective-binding substance is immobilized, and a fiber array
comprising a bundle of the fibers are provided. By the present
invention, a fiber on which a selective binding substance is
immobilized efficiently with a good reproducibility is provided.
Further, by combining these fibers to form a fiber array, a
selective binding substance-immobilized fiber array in which the
selective binding substances are arrayed arbitrarily and accurately
with a high density may be efficiently obtained. Further, by using
optical fibers, the bound sample may be detected through the fiber
simply and efficiently. Further, by labeling each fiber, each fiber
to which the test substance is bound may be identified without
relying on the position of the fiber in the array.
[0023] Further, by the method for selective binding reaction, and
the apparatus and substrate therefor, the efficiency of the
selective binding reaction is promoted, so that the selective
binding reaction step may be completed in a short time even when
the concentration of the corresponding selective binding substance
in the test sample is low.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 shows a schematic cross-sectional view and plan view
of the apparatus for selective binding reaction according to the
present invention;
[0025] FIG. 2 is a drawing showing the principle of the behaviors
of the selective binding substances in the method for selective
binding reaction according to the present invention;
[0026] FIG. 3 is a schematic cross-sectional view of the
conventional hybridization apparatus;
[0027] FIG. 4 is a drawing showing the principle of the behaviors
of the selective binding substances in the method for selective
binding reaction according to the present invention; and
[0028] FIG. 5 is a schematic view of the optical parts in the
measuring apparatus used in Example 5.
BEST MODE FOR CARRYING OUT THE INVENTION
[0029] In the present specification and claims, the term "selective
binding substance" means a substance which can selectively bind to
a test substance directly or indirectly. Representative examples
thereof include nucleic acids, proteins, saccharides, and other
antigenic compounds. The nucleic acid may be a DNA or RNA. Since a
single-stranded nucleic acid having a particular nucleotide
sequence selectively hybridizes with a single-stranded nucleic acid
having a nucleotide sequence complementary to the nucleotide
sequence of the nucleic acid or a part thereof, the single-stranded
nucleic acid is a "selective binding substance" referred to in the
present invention. Examples of the proteins include antibodies,
antigen-binding fragments of antibodies such as Fab fragment and
F(ab').sub.2 fragment, and various antigens. Since an antibody or a
antigen-binding fragment selectively binds to the corresponding
antigen, and since an antigen selectively binds to the
corresponding antibody, they are "selective binding substances". As
the saccharides, polysaccharides are preferred, and examples
thereof include various antigens. Antigenic substances other than
proteins and saccharides may also be immobilized. Especially
preferred "selective binding substances" are nucleic acids,
antibodies and antigens. The selective binding substance used in
the present invention may be a commercially available product or
one obtained from living cells or the like. The term "corresponding
selective binding substance" herein means a substance which
selectively binds to the above-mentioned selective binding
substance. For example, in cases where the selective binding
substance is a single-stranded nucleic acid, the single-stranded
nucleic acid having the nucleotide sequence complementary to that
of the single-stranded nucleic acid is the corresponding selective
binding substance; and in cases where the selective binding
substance is an antibody or an antigen-binding fragment thereof,
the antigen or hapten which undergoes the antigen-antibody reaction
with the antibody or an antigen-binding fragment thereof, is the
corresponding selective binding substance.
[0030] Examples of the fibers which may be used for the
immobilization of the selective binding substances include chemical
fibers such as synthetic fibers, semi synthetic fibers, regenerated
fibers and inorganic fibers, natural fibers and composite fibers of
these.
[0031] Representative examples of synthetic fibers include various
polyamide fibers such as nylon 6, nylon 66 and aromatic polyamides;
various polyester fibers such as polyethylene terephthalate,
polybutylene terephthalate, polylactic acid and polyglycolic acid
fibers; various acrylic fibers such as polyacrylonitrile fibers;
various polyolefin fibers such as polyethylene and polypropylene
fibers; various polyvinyl alcohol fibers; various polyvinylidene
fibers; polyvinyl chloride fibers; various polyurethane fibers;
phenol fibers; fluorine-contained fibers such as
polyfluorovinylidene and polytetrafluoroethylene fibers; and
polyalkylene p-oxybenzoate fibers. Fibers other than those for
clothes, for example, optical fibers mainly composed of transparent
amorphous macromolecules such as polymethyl methacrylate and
polystyrene may also be employed. Especially preferred are plastic
optical fibers of which cores are composed of a material such as
polymethyl methacrylate, polystyrene or polycarbonate, and of which
clad is composed of a plastic having a smaller refractive index
than the core. Either the so called sheath/core fibers or graded
index fibers may be employed. Further, coated plastic optical
fibers may be employed. As the coating material, thermoplastic
resins such as polyethylenes, PVC, urethanes and fluorine-contained
resins, and various rubber tubes may be employed.
[0032] Representative examples of semi synthetic fibers include
various cellulose derivative fibers derived from diacetate,
triacetate, chitin or chitosan; and various protein-based fibers
called promix. Representative examples of regenerated fibers
include various cellulose-based regenerated fibers (rayon, cupra,
polynosic and the like) obtained by viscose process, cuprammonium
process or organic solvent process.
[0033] Representative examples of the inorganic fibers include
glass fibers, carbon fibers and metal fibers such as Au, Ag, Cu and
Al fibers. Especially preferred are optical fibers made of a
light-transmitting glass. Similar to the plastic optical fibers,
either the so called sheath/core fibers or graded index fibers may
be employed. Further, coated plastic optical fibers may be
employed. Glass-plastic composite fibers may also be employed.
[0034] Representative examples of natural fibers include plant
fibers such as cotton, flax, ramie and jute fibers; animal fibers
such as wool and silk fibers; and mineral fibers such as asbestos
fibers. The form of the fiber used in the present invention is not
restricted. It may be a monofilament or multifilament. Further,
spun yarn obtained by spinning short fibers may also be employed.
When using a fiber in the form of multifilament or yarn, the
selective binding substance may be immobilized in the spaces among
the filaments or the like.
[0035] The fiber used in the present invention may be a non-treated
fiber, or may be one to which reactive functional groups are
introduced, or may be one subjected to plasma treatment or
irradiation treatment with y-ray, electron beam or the like.
Immobilization of the selective binding substance on these fibers
may be attained by known methods utilizing various chemical or
physical interactions between the fiber and the selective binding
substance, that is, chemical or physical interactions between the
functional groups on the fiber and the selective binding
substance.
[0036] In cases where the same selective binding substance is
immobilized, a plurality of fibers may be treated in one batch.
Although selective binding substance may be immobilized on the
entire fiber, it is preferred to immobilize the selective binding
substance on an end region of the fiber. The end region may be the
end face of the fiber and/or the side face in the vicinity of the
end face. To distinguish the fibers on which different selective
binding substances are immobilized, each fiber may be fixed to a
support, thereby distinguishing the fibers on which different
selective binding substances are immobilized. These support may be
separate or connected each other. Alternatively, fibers having
different shapes may be employed.
[0037] The fibers immobilizing different samples may also be
distinguished by coloring the fibers and distinguishing the fibers
based on the wavelengths of the colors. A method in which the light
emitted to the outside from the fiber is detected, or a method in
which a light passing through the fiber is guided to a
photodetector, thereby distinguishing the fibers, may also be
employed.
[0038] The fibers may also be distinguished by using fibers on
which characters or marks are recorded. For example, bar code or
the like is recorded on or in the fiber, and the fibers
immobilizing different samples may be distinguished by reading the
bar code or the like.
[0039] Electroconductivity may be given to the fibers by
incorporating an electroconductive material such as a metal,
carbon, electroconductive polymer or a magnetic material in the
fibers, or by coating the fibers with a material such as a metal by
a method such as sputtering, vapor deposition, plating, CVD or the
like. A material belonging to 3A group to 5B group in the second
period to fourth period in the periodic table or a mixture thereof
may be applied by the above-mentioned method. By so doing, fibers
immobilizing different samples may be distinguished electrically or
magnetically.
[0040] As the fibers used in the present invention, thin fibers are
preferred. In the preferred mode of the present invention, the
thickness of one fiber is not larger than 1 mm. As for
monofilaments, commercially available fishing lines have
thicknesses of 50 to 900 .mu.m. By the recent spinning technique, a
monofilament having a thickness of 1 dtex (in case polyethylene
terephthalate, about 8 .mu.m diameter) may be produced, and even
thinner fiber (extra fine fiber or ultra extra thin fiber) (1 to 10
.mu.m diameter) may be produced. In case of using an optical fiber,
the filament preferably has a diameter of 3 to 1000 .mu.m, more
preferably about 50 to 1000 .mu.m. In view of ease of handling,
plastic optical fibers, glass optical fibers and composite optical
fibers made of a glass and resin, which have diameters of about 50
to 500 .mu.m are preferred. A so called image conduit, which is a
bundle of very thin optical fibers having diameters of 2 to 50
.mu.m may also preferably be employed. The measurement may be
carried out by immobilizing each selective binding substance on
each image conduit, and by collecting the image conduits. In cases
where the binding state is detected by light, by using this image
conduit, the converged light does not diverge in the optical fiber,
so that detection light may be efficiently transmitted to the
sample. The number of optical fibers contained in the image conduit
is preferably 2 to 50,000. If it is more than 50,000, the outer
diameter of the image conduit is too large, and handling thereof
may be troublesome in some cases.
[0041] In cases where an optical fiber is used, by employing a
light-emitting label such as fluorescent label or a
color-generating label as the label attached to the test substance
hereinbelow described in detail, the binding of the test substance
may be measured by measuring the light which passes through the
optical fiber and emitted from another end of the optical fiber. In
this case, it is also possible to identify which fiber is lighting.
This is preferred, and this is advantageous for automation of the
apparatus. In cases where an electroconductive fiber is used, the
binding of the test substance may be measured as an electric signal
at the other end of the fiber. In this case too, it is also
possible to identify the fiber to which the test substance is
bound, that is preferred and that is advantageous for automation of
the apparatus. Further, by using an electroconductive fiber, the
binding may be accelerated by using means for applying electric
field or electric current. Especially, in case of accelerating the
hybridization between nucleic acids, it is preferred to
substantially use the fiber on which the nucleic acid is
immobilized as an anode. The arrangement of a cathode is not
restricted as long as the cathode does not directly contact the
anode. Although the type of electric field applied is preferably
direct current, alternating voltage may also be employed under a
condition in which the fiber substantially acts as an anode. That
is, even in cases where alternating electric field is applied, it
is acceptable as long as the fiber substantially acts as an anode
by making the time periods for applying positive voltage be longer
than the time period for applying negative voltage, by making the
absolute value of the positive voltage be larger than that of the
negative voltage, or by combination of these. Although the voltage
applied is not restricted, not less than 0.1V and not more than
5000V is preferred. If the voltage applied is less than 0.1V, the
effect by applying electric voltage may not be obtained
sufficiently in some cases, and if it is higher than 5000V,
handling may be difficult. Whether electric current flows or not
when the voltage is applied is not restricted. When the cathode is
arranged such that it does not contact the reaction solution,
electric current does not flow, and if the cathode is arranged such
that it contacts the reaction solution, electric current flows. In
cases where electric current flows, 1 to 2000 mA is preferred.
[0042] On the other hand, as mentioned above, the present invention
also provides a method for binding reaction between a selective
binding substance and a corresponding selective binding substance,
comprising immobilizing the selective binding substance on a
substrate; and making a test sample contacted to the immobilized
selective binding substance, which test sample contains the
corresponding selective binding substance which selectively binds
to the selective binding substance, thereby carrying out the
binding reaction; the test sample and/or the corresponding
selective binding substance being moved relative to the surface on
which the selective binding substance is immobilized, in the step
of immobilizing the selective binding substance on a substrate and
making a test sample contacted to the immobilized selective binding
substance. The phrase "moved relative to" means that the
corresponding selective binding substance is moved relative to the
surface immobilizing the selective binding substance when the
surface immobilizing the selective binding substance is viewed
laterally.
[0043] In this method for binding reaction, although it is
preferred to use the above-described fiber on which the selective
binding substance is immobilized or the fiber array, this is not
indispensable, and the method may be applied to the methods
utilizing the conventional microarrays, microplates and the
like.
[0044] As the method for moving the corresponding selective binding
substance in the vicinity of the selective binding substance
immobilized on the microarray substrate, fiber or the like so as to
increase the probability of collision between the selective binding
substance and the corresponding selective binding substance, a
method in which electric field is formed so as to move the
electrically charged selective binding substance; a method in which
a channel is formed in the substrate immobilizing the selective
binding substance, and the test sample is moved using a micro pump;
a method in which a function like a magnetic fluid is given to the
test sample, and the test sample is moved by an external physical
force; and a method in which the substrate immobilizing the
selective binding substance is moved, and the like are thought.
[0045] Among these, the electric field-applying method which has an
effect for increasing the probability of collision by the simplest
structure, especially the method in which alternating electric
field is applied is preferred. In general, nucleic acids are
negatively charged in aqueous solutions. Further, macromolecules
such as proteins and polysaccharides are usually also electrically
charged in aqueous solutions. Therefore, by applying electric field
to a reaction liquid, it is possible to move the corresponding
selective binding substance. Particularly, by applying alternating
electric field, it is possible to reciprocally move the
corresponding selective binding substance, so that the probability
of collision with the selective binding substance may be
efficiently increased.
[0046] Thus, as a preferred mode for binding reaction according to
the present invention, a method for binding reaction is provided
wherein the binding reaction is carried out while applying an AC
voltage in a direction crossing the perpendicular axis of the
surface on which the selective binding substance is immobilized,
between electrodes arranged outside the both ends of the area on
which the selective binding substance is immobilized, in the step
of immobilizing the selective binding substance on a substrate and
making a test sample contacted to the immobilized selective binding
substance, which test sample contains the corresponding selective
binding substance which selectively binds to the selective binding
substance, thereby carrying out the binding reaction.
[0047] The perpendicular axis of the surface on which the selective
binding substance is immobilized is, for example, the dot-and-dash
line A shown in FIG. 1(a), that is, it means the direction
perpendicular to the surface on which the selective binding
substance is immobilized. The direction crossing this perpendicular
axis means a direction which crosses the above-mentioned
perpendicular axis when the surface immobilizing the selective
binding substance is viewed laterally. The "direction crossing the
perpendicular axis" is preferably the direction perpendicular to
the perpendicular axis as shown in FIG. 2. However, the direction
is not necessarily the one perpendicular to the perpendicular axis,
and excellent effects may also be obtained when the angle formed by
the line which connect the opposing electrodes through the shortest
distance and the direction perpendicular to the perpendicular axis
is not more than 45.degree., preferably not more than
30.degree..
[0048] Although the voltage applied is not restricted, if the
voltage is too low, the effect obtained by applying electric field
is small, and, on the other hand, if it is too high, the selective
binding substance and/or the corresponding selective binding
substance may be damaged. Thus, the voltage is preferably about 5V
to 50V per 1 cm of the distance between the electrodes, more
preferably about 10V to 25V. The frequency of the alternating
voltage is not restricted, and a frequency may preferably be about
1 Hz to 100 Hz, more preferably about 5 Hz to 20 Hz.
[0049] The number of sites immobilizing the selective binding
substance may be one (e.g., one well in a microplate). However, if
there are a plurality of sites immobilizing selective binding
substance exist, and a selective binding substance-arrayed region
(e.g., the region denoted by reference numeral 8 in FIG. 1(a)
mentioned below) in which the selective binding
substance-immobilizing sites are arrayed exists, measurement of a
plurality of types of substances may be measured simultaneously in
parallel, so that it is preferred. In this case, the electrodes are
preferably arranged outside the both ends of the selective binding
substance-arrayed region.
[0050] The method for binding reaction by the above-described
electric field-applying method may be carried out by using a
substrate for conducting a binding reaction including a step of
immobilizing the selective binding substance on the substrate and
making a test sample contacted to the immobilized selective binding
substance, which test sample contains the corresponding selective
binding substance that selectively binds to the selective binding
substance, thereby carrying out the binding reaction, the substrate
comprising a site for immobilizing the selective binding substance;
and electrodes arranged outside the both ends of the area on which
the selective binding substance is immobilized. On the "site for
immobilizing the selective binding substance", a selective binding
substance is immobilized, and this immobilization may be carried
out by an end user before use, or the manufacturer of the substrate
may produce and sell one on which the selective binding substance
for a particular test is immobilized beforehand.
[0051] In the method for selective binding reaction or the
apparatus for selective binding reaction, examples of the materials
which may be used in the electrodes include simple metals such as
platinum, gold, silver, chromium, titanium, nickel, aluminum,
copper and palladium, oxides and nitrides of these metals, alloys
thereof, carbon and carbon compounds, and electrically conductive
polymers, and it is acceptable if at least one selected from these
is contained.
[0052] As for the characteristics of the simple metals, oxides and
nitrides thereof, and alloys thereof, since electric current flows
between the electrodes through the test sample containing
corresponding selective binding substance when AC voltage is
applied between the electrodes arranged using these materials, the
materials which hardly react with the test sample and which hardly
elute metal ions into the test sample are preferred.
[0053] Representative examples of the carbon compounds include
graphite and fullerene.
[0054] Representative examples of electroconductive polymers
include polyacetylene, polypyrrol, polythiophine and polyaniline.
Composite electroconductive plastics having improved
electroconductive properties obtained by mixing the above-mentioned
electroconductive polymer and the above-mentioned metal, carbon
compound or the like may also be exemplified.
[0055] Although it is preferred to form the electrodes beforehand
on the substrate for immobilizing the selective binding substance
because of the reason described below, the mode wherein the
electrodes are arranged on the side of the apparatus for binding
reaction, and wherein the electrodes are mounted on the substrate
for immobilizing the selective binding substance during the step of
preparation for the binding reaction is also acceptable.
[0056] Methods for forming the electrodes on the substrate using a
metal include a method in which a mask with openings having the
shapes of the electrodes is mounted on the substrate, and the
electrodes are formed by sputtering method or vapor deposition
method; a method in which electrodes in the form of thick films are
formed by plating method; and a method in which metal foils or
metal thin plates are adhered to the substrate by an adhesive. In
case of using a carbon compound, the electrodes may be prepared by
mounting a mask with openings having the shapes of the electrodes,
and forming the electrodes by sputtering method. In case of using
an electroconductive polymer, the electrodes may be formed by
applying an electroconductive polymer in the form of paste by a
printing method such as silk screen printing, and by curing the
paste by photo-curing by UV light.
[0057] In cases where the electrode are retained in the apparatus
for selective binding reaction, electrode plates in the form of a
thin plate prepared by using the above-mentioned metal material,
carbon compound or the electroconductive polymer are arranged on
the base of the apparatus for selective binding reaction, and the
electrode plates are mounted on the substrate for immobilizing the
selective binding substance after mounting the substrate for
immobilizing the selective binding substance on the base, thereby
forming the electrodes.
[0058] A mode of the present invention will now be described
referring to the drawings. The side view and the plan view of a
mode of the apparatus for selective 10 binding reaction according
to the present invention are shown in FIG. 1(a) and FIG. 1(b),
respectively. It should be noted that the present invention is not
limited to this example. As shown in FIGS. 1 and 2, the apparatus
comprises a substrate 1 on which selective binding substances are
arrayed, electrodes 2 and 3, a cover plate 5 and means for applying
AC voltage 6. Selective binding substances 10 are immobilized on
the sites 4 for immobilizing selective binding substance arranged
on the selective binding substance-arraying substrate 1, thereby
forming a selective binding substance-arrayed region 8. On the
selective binding substance-arraying substrate 1 mounted on a base
9, electrodes 2 and 3 are arranged on both sides of the selective
binding substance-arrayed region 8, and a cover plate 5 bridging
the electrodes 2 and 3 is mounted on the electrodes 2 and 3. The
space sandwiched between the selective binding substance-arraying
substrate 1 and the cover plate 5 via electrodes 2 and 3 is filled
with a test sample solution 7 containing corresponding selective
binding substances 11.
[0059] Although it is preferred to array the selective binding
substances 10 on the lattice points of a two-dimensional lattice in
the selective binding substance-arrayed region 8, they may be
arranged on the positions shifted from the lattice points, or may
be arranged along a straight line. Alternatively, the sites 4 for
immobilizing selective binding substance may be arranged
three-dimensionally in the direction perpendicular to the surface
of the selective binding substance-arraying substrate 1 such that
they form steps.
[0060] After filling the above-mentioned space with the test sample
solution 7, a voltage is applied between the electrodes 2 and 3
using the means for applying AC voltage 6. By this, an electric
field is generated between the electrodes 2 and 3. Since the
corresponding selective binding substances 11 naturally diffused in
the test sample solution 7 have negative charges, they repeat
movement in the direction crossing the selective binding
substance-arrayed region 8 in accordance with the direction of the
electric field generated between the electrodes 2 and 3. More
particularly, when the electrode 2 is positively biased and
electrode 3 is negatively biased, the corresponding selective
binding substances 11 are attracted to the electrode 2, and when
the electrode 2 is negatively biased and electrode 3 is positively
biased, the corresponding selective binding substances 11 are
attracted to the electrode 3. During the movement of the
corresponding selective binding substances crossing the selective
binding substance-arrayed region 8, the corresponding selective
binding substances 11 contact the selective binding substances 10
immobilized on the sites 4 for immobilizing the selective binding
substance, so as to attain hybridization when they have
complementary sequences.
[0061] The higher the voltage applied between the electrodes, the
larger the electric attracting force and the electric repulsive
force exerted to the corresponding selective binding substances 11
by the electrodes, and so the higher the effects of the contact of
the corresponding selective binding substances 11 with the
selective binding substances 7 due to the movement of the
corresponding selective binding substances 11. However, if a high
voltage is applied for a long time, the selective binding
substances 7 and the corresponding selective binding substances 11
may be damaged. Therefore, in this mode, the voltage per 1 cm of
the distance between the electrodes is 5V to 50V, and a voltage of
10V to 25V per 1 cm of the distance between the electrodes is more
preferred in order to attain stable hybridization.
[0062] As explained above, in the method for promoting efficiency
of selective binding reaction by using AC electric field according
to the present invention, the selective binding substances 10 and
the corresponding selective binding substances 11 are always moved
relatively during the binding reaction, and so repeat collision and
contact therebetween, the efficiency of the binding reaction is
promoted.
[0063] As the sites for immobilizing the selective binding
substance, areas in a surface on the substrate or a part of the
substrate, hollow or protruded regions formed on the substrate are
usually employed. Alternatively, rod-shaped resin, glass, metal,
fibers or the like are inserted into holes penetrating the
selective binding substance-arraying substrate 1, and the ends of
the resin, glass, metal and fibers may be used as the sites for
immobilizing the selective binding substance. Especially, by using
the end faces of the above-described fibers or fiber bundle
according to the present invention as the sites for immobilizing
the selective binding substance, detection at the other end of the
each of the fibers may be attained, so that it is preferred.
[0064] To reduce the amount of the valuable test sample solution 7,
the thickness of the electrodes 2 and 3 is preferably as small as
possible, more preferably 5 .mu.m to 200 .mu.m. To form such very
thin electrodes of which irregularities in the thickness are small,
it is preferred to form the electrodes on the selective binding
substance-arraying substrate beforehand. However, it is also
acceptable to provide the electrodes on the side of the apparatus
for binding reaction, and mounting the electrodes on the selective
binding substance-arraying substrate 1 during the step of
preparation for the binding reaction.
[0065] In the conventional methods, since the hybridization
reaction depends on the natural diffusion of the selective binding
substances, the probability of contact between the selective
binding substances 10 and the corresponding selective binding
substances 11 is low, so that the efficiency of hybridization
reaction is low. Further, in the methods which tried to overcome
this inefficiency by utilizing electric attraction as shown in
FIGS. 3 and 4, the promotion of the efficiency is not
sufficient.
[0066] The conventional methods for promoting hybridization
efficiency utilizing electric attraction will be described
referring to FIGS. 3 and 4. The selective binding substances 10 are
immobilized on sites 15 for immobilizing selective binding
substance formed on a selective binding substance-arraying
substrate 12. On the selective binding substance-arraying substrate
12 and outside the region in which the selective binding substances
10 are arrayed, supports 13 are arranged, and a cover plate 14
bridging the supports 13 is mounted. The space sandwiched between
the selective binding substance-arraying substrate 12 and the cover
plate 14 via the supports 13 is filled with a test sample solution
18. After filling the space with the test sample solution 18, an
electrode 16 is negatively biased, and the sites 15 for
immobilizing selective binding substance having an
electroconductive layer is positively biased using means for
applying electric voltage 17. As a result, an electric field is
generated between the electrode 16 and the sites 15 for
immobilizing selective binding substance. Since the corresponding
selective binding substances 11 naturally diffused in the test
sample solution 18 have negative charges, they are attracted to the
sites 15 for immobilizing selective binding substance. As a result,
the concentrations of the corresponding selective binding
substances 11 in the vicinity of the sites 15 for immobilizing
selective binding substance are increased, so that the selective
binding substance 10 and the corresponding selective binding
substance 11 contact during the process of adsorption of the
corresponding selective binding substances 11.
[0067] However, by the method shown in FIG. 3, the corresponding
selective binding substances 11 are adsorbed on the sites 15 for
immobilizing selective binding substance by the electric force, and
the selective binding substances 10 having negative charges similar
to the corresponding selective binding substances 11 are also
adsorbed on the surfaces of the selective binding
substance-immobilizing sites 15, so that the relative movements
between the selective binding substance 10 and the corresponding
selective binding substance 11 become small. Therefore, the
probability of contact between the selective binding substances 10
and the corresponding selective binding substance 11 is smaller
than in the method according to the present invention.
[0068] The DNA or RNA used as the selective binding substance or
corresponding selective binding substance may be those prepared
from living cells or may be those chemically synthesized.
Preparation of the DNA or RNA from living cells may be carried out
by known methods. For example, extraction of DNA may be carried out
by the method of Blin et al., (( Blin et al., Nucleic Acids Res. 3:
2303 (1976)), and extraction of RNA may be carried out by the
method of Favaloro et al., (Favaloro et al., Methods Enzymol.65:
718 (1980)). The nucleic acids to be immobilized may also be linear
or circular plasmid DNAs and chromosomal DNAs, DNA fragments
obtained by cleaving these DNAs by a restriction enzyme or obtained
by chemically cleaving these DNAs, synthetic DNAs prepared in vitro
by using an enzyme or the like, chemically synthesized
oligonucleotides or the like.
[0069] On one fiber or on each site for immobilizing selective
binding substance, one type of selective binding substance is
usually immobilized. However, a plurality of types of selective
binding substances may be immobilized on one fiber or one site for
immobilizing selective binding substance in cases, for example,
where a plurality of types of genes having mutations are desired to
be immobilized on the same site for immobilization.
[0070] As the selective binding substance immobilized on a
plurality of fibers or on a plurality of sites for immobilizing
selective binding substance, different types of selective binding
substances may be immobilized on the different fibers or the
different sites, or the same type of selective binding substance
may be immobilized on the different fibers or the different sites.
Alternatively, one type of selective binding substance may be
immobilized on a part of the fibers or a part of the sites for
immobilizing selective binding substance, and another type of
selective binding substance may be immobilized on a plurality of
other fibers or other sites for immobilizing selective binding
substance. The types of the selective binding substances and order
of arrangement are not limited by the positions of the fibers in
the fiber array. It is also effective to immobilize the same
selective binding substance on a plurality of fibers or a plurality
of sites for immobilizing selective binding substance, thereby
increasing the measurement sensitivity.
[0071] Immobilization of the selective binding substance on the
support fiber or on the site for immobilizing selective binding
substance may be carried out by a known method. In cases where an
unmodified selective binding substance is immobilized on the fiber
or on the site for immobilizing selective binding substance, the
selective binding substance may be immobilized by making the
selective binding substance contact the fiber or the site for
immobilizing selective binding substance, and then subjecting the
resultant to baking or UV irradiation. In the Examples below, DNAs
are immobilized on polymethyl methacrylate optical fibers or
polymethyl methacrylate substrate by this method. In cases where a
selective binding substance modified with amino groups is to be
immobilized on a fiber, the selective binding substance may be
bound to a functional group in the fiber using a crosslinking agent
such as glutaraldehyde or
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).
[0072] The temperature at which the sample containing the selective
binding substance is made to contact the fiber is preferably
5.degree. C. to 95.degree. C., more preferably 15.degree. C. to
65.degree. C. The treatment time is usually 5 minutes to 24 hours,
and preferably not less than 1 hour.
[0073] In the present invention, the selective binding substance
may be immobilized on the fiber or the site for immobilizing
selective binding substance as it is. Alternatively, a derivative
of the selective binding substance obtained by chemical
modification, or a denatured nucleic acid may be immobilized as
required. Known chemical modification of nucleic acids include
amination, biotinylation and attachment of digoxigenin [Current
Protocols In Molecular Biology, Ed.; Frederick M. Ausubel et
al.(1990); and (Non-Isotope Experimental Protocol (1) DIG
Hybridization (Shujunsha)], and these modification methods may be
employed in the present invention. As an example, introduction of
amino groups into a nucleic acid will now be described. The
positions at which the aliphatic hydrocarbon chain having an amino
group and the nucleic acid are bound are not restricted, and may be
not only the 5'-end or 3'-end of the nucleic acid, but also may be
within the nucleic acid chain (e.g., phosphoric acid diester moiety
or base moiety). This single-stranded nucleic acid derivative may
be prepared in accordance with methods described in Japanese
Laid-open Patent Application (Kokai) No. 3-74239, U.S. Pat. No.
4,667,025 and U.S. Pat. No. 4,789,737. Other than this method, the
amino group may be introduced by, for example, using a commercially
available reagent for introducing amino groups [e.g., Aminolink II
(trademark), PE BIOSYSTEMS JAPAN, or Amino Modifiers (trademark),
CLONETECH], or by a well-known method (Nucleic Acids
Res.,11(18),6513-(1983) ) for introducing amino group into the
phosphate at the 5'-end of the DNA.
[0074] The fiber immobilizing the selective binding substance or
the substrate immobilizing the selective binding substance may be
subjected to an appropriate treatment after immobilizing the
selective binding substance. For example, the immobilized selective
binding substance may be denatured by heat treatment or by
treatment with an alkali or a surfactant. In case of using a
selective binding substance obtained from a biological material
such as a cell or bacterium, unnecessary cellular components may be
removed. The fiber or the substrate for immobilizing selective
binding substance after the treatment may be used as a material for
detecting the selective binding substance. These treatments may be
performed separately or simultaneously. Alternatively, the
treatment may be performed before a sample containing the selective
binding substance is immobilized on the fiber or the substrate for
immobilizing selective binding substance.
[0075] By making the fiber or the substrate for immobilizing
selective binding substance according to the present invention on
which the selective binding substance is immobilized as a probe
interact with a test substance, the particular test substance in
the sample may be detected. Labeling (so as to attain
distinguishment) described below may be performed on two types of
test samples, and the difference may be compared.
[0076] For the detection of the corresponding selective binding
substance in the test sample, which selectively binds to the
selective binding substance, known means for specifically
recognizing the binding may be employed. For example, a label such
as a fluorescent substance, luminescent substance, radio isotope is
bound to the corresponding selective binding substance in the
sample, and after the selective binding reaction and washing, the
label may be detected. The type of the label and the method for
introducing the label are not restricted at all, and various known
means may be employed. Particularly, in cases where the fiber
transmits light, and the labeled test substance is subjected to the
reaction at an end of the fiber, and the label is detected with a
detector at the other end of the fiber, it is preferred to use a
fluorescent substance or luminescent substance as the label.
Fluorescent substances and luminescent substances used for
immunoassays and for the measurements of hybridization of nucleic
acids are well-known in the art and various such substances are
commercially available. These commercially available fluorescent
substances and luminescent substances may be employed.
[0077] The measurement of the result of the binding reaction may
also be carried out by reacting a free labeled substance for
measurement, which selectively binds to the corresponding selective
binding substance after or simultaneously with the binding reaction
between the selective binding substance immobilized on the fiber or
on the site for immobilizing the selective binding substance and
the corresponding selective binding substance in the sample, and by
measuring the label of the substance for measurement immobilized on
the fiber via the corresponding selective binding substance and the
selective binding substance after washing. For example, in a case
where a nucleic acid having a particular nucleotide sequence as a
selective binding substance is immobilized on a fiber, and the
corresponding selective binding substance is a nucleic acid
containing a region complementary to the nucleic acid, a nucleic
acid complementary to a region in the nucleic acid which is the
corresponding selective binding substance, other than the region
complementary to the selective binding substance, may be used as
the substance for measurement after labeling. Similarly, in a case
where an antigen as the selective binding substance is immobilized
on a fiber, and the corresponding selective binding substance is an
antibody which undergoes antigen-antibody reaction with the
antigen, a labeled secondary antibody which undergoes
antigen-antibody reaction with the antibody may be used as the
substance for measurement. In these cases, it is preferred to
immobilize the selective binding substance on one end region of the
fiber, to employ a fluorescent or luminescent substance as the
label, and to measure the result of the reaction from the other end
of the fiber.
[0078] In cases where the fiber or the site for immobilizing
selective binding substance is electrically conductive, it is
preferred to employ a method for detecting a response such as
electric current based on an electrochemical reaction. In this
case, existence or degree of binding between the selective binding
substance and the test sample may be detected by reacting the
selective binding substance immobilized on the fiber acting as an
electrode and the test sample in the presence of a material which
accelerates or suppresses the reaction between the selective
binding substance and the test sample, wherein all or a part of the
material is contained in the bound selective binding substance and
test sample, and by measuring the electric current flowing the
electrode, that is, the fiber or the site for immobilizing the
selective binding substance after the reaction.
[0079] Examples of the test substances subjected to the measuring
method using the fiber or fiber array according to the present
invention, or to a measuring method using the substrate for
immobilizing selective binding substance applied to the apparatus
for binding reaction according to the present invention include
genes of pathogenic bacteria, viruses and the like, causative genes
of hereditary diseases and. parts thereof, various biological
components having antigenecities, and antibodies to pathogenic
bacteria, viruses and the like, although the test substances are
not restricted thereto. Examples of the samples containing such
test substances include body fluids such as blood, serum, plasma,
urine, feces, spinal fluid, saliva and various tissue fluids;
various foods and beverages as well as dilutions thereof. The
nucleic acid which is a test substance may be a labeled nucleic
acid extracted from blood or cells by a conventional method and
labeled, or may be an amplified nucleic acid obtained by a nucleic
acid-amplification method such as PCR. In the latter case, the
measurement sensitivity may be largely promoted. In cases where an
amplification product of a gene is used as the test substance, by
carrying out the amplification in the presence of a nucleoside
triphosphate labeled with a fluorescent substance or the like, the
amplified nucleic acid can be labeled. In cases where the test
substance is an antigen or an antibody, the antigen or antibody
which is the test substance may be directly labeled by a
conventional method. Alternatively, after binding the antigen or
antibody which is the test substance with the selective binding
substance, the fiber or the site for immobilizing selective binding
substance on which the selective binding substance has been
immobilized is washed, reacting a labeled antibody or antigen which
undergoes antigen-antibody reaction with the antigen or antibody,
and the amount of the label bound to the fiber or to the site for
immobilizing selective binding substance is measured.
[0080] The step of interacting the immobilized substance and the
test substance may be carried out in exactly the same manner as in
the conventional method. The reaction temperature and the time are
appropriately selected depending on the length of the chain of the
nucleic acids to be hybridized, type of the antigen and/or antibody
involved in the immune reaction. Usually, in case of hybridization
of nucleic acids, the reaction may be carried out at about
50.degree. C. to 70.degree. C. for 1 minute to several hours, and
in case of immune reaction, the reaction may be carried out at room
temperature to about 40.degree. C. for 1 minute to several
hours.
[0081] By the above-described method, the amount of the test
substance such as nucleic acid, antibody or antigen, which
selectively binds to the immobilized selective binding substance
may be measured. That is, in cases where a nucleic acid is
immobilized as the selective binding substance, the amount of a
nucleic acid having a sequence complementary to the immobilized
nucleic acid or a part thereof may be measured. In cases where an
antibody or antigen is immobilized as the selective binding
substance, the amount of the antigen or antibody which undergoes
immune reaction with the immobilized antibody or antigen may be
measured. It should be noted that the term "measure" used in the
present specification include both detection and
quantification.
[0082] By using the present invention, expression of genes,
proteins and sugar chains in various organisms may be investigated
efficiently, quickly and simply. For example, after labeling the
nucleic acids extracted from a normal human liver and a liver
infected with a hepatitis virus, the nucleic acids are subjected to
hybridization with each of the various known human genes
immobilized on the fibers or on the substrate for immobilizing
selective binding substance according to the present invention. By
comparing the degree of binding of the nucleic acids extracted from
the normal and hepatitis-infected livers, the change in the gene
expression in the hepatitis liver may be investigated.
[0083] Similarly, by binding the proteins extracted from a normal
brain and from a brain suffering from Alzheimer's disease are
subjected to a binding reaction with a fiber array on which various
monoclonal antibodies which are proteins are immobilized, and by
comparing the bound proteins between the normal and Alzheimer's
brains, abnormal expression of proteins in Alzheimer's brain may be
investigated.
EXAMPLES
[0084] The present invention will now be described in more detail
by way of examples below. However, the present invention is not
limited to the examples below.
Example 1
[0085] To confirm that immobilization of nucleic acids on the fiber
or glass substrate used in this example and hybridization thereof
can be carried out surely, experiments were carried out using
digoxigenin as a label, which does not cross-react with biological
samples, and which is thermally stable and is not decomposed by the
heat given when the hybridization is carried out.
[0086] One end of each of polymethyl methacrylate optical fibers
was dipped in a nucleic acid solution of actin gene (produced by
Takara Shuzo Co., Ltd.) (concentration of the nucleic acid: 10
.mu.g/ml), and the resultant was subjected to UV treatment (UV
Crosslinker produced by Stratagene) after drying in the air, to
obtain fibers on which the nucleic acid was immobilized. An
oligonucleotide having a nucleotide sequence complementary to a
part of the sequence of the used nucleic acid was synthesized, and
labeled with digoxigenin (DIG, Roche Diagnostics).
[0087] On the other hand, the nucleic acid solution of actin gene
(produced by Takara Shuzo Co., Ltd.) (concentration of the nucleic
acid: 10 .mu.g/ml) was spotted on a slide glass to which amino
groups had been introduced, such that each size of the immobilized
site had a diameter of about 200 .mu.m, and the resultant was
subjected to UV treatment (UV Crosslinker produced by Stratagene)
after drying in the air, to obtain a substrate on which the nucleic
acid was immobilized. The oligonucleotide having a nucleotide
sequence complementary to a part of the sequence of the used
nucleic acid was synthesized, and labeled with digoxigenin (DIG,
Roche Diagnostics).
[0088] Each oligonucleotide of which end was aminated was dissolved
in 100 mM boric acid buffer (pH8.5) to a final concentration of 2
mM. Equal amount of
digoxigenin-3-O-methylcarbonyl-.alpha.-aminocaproic
acid-N-hydroxy-succinimide ester (26 mg/ml solution in
dimethylformamide) was added to the solution, and the resulting
mixture was left to stand overnight at room temperature. Ethanol
precipitation was performed using glycogen (Roche Diagnostics) as a
carrier, and the precipitates were dissolved in 100 .mu.mol of 10
mM Tris-HCl (pH7.5) and 1 mM EDTA after drying in the air. The thus
obtained DIG-labeled oligonucleotide was used as a model of the
sample nucleic acid.
[0089] The prepared nucleic acid-immobilized fibers were placed in
a bag for hybridization, and hybridization was carried out by a
conventional method (in accordance with the instructions in the
manual of the product of Roche Diagnostics).
[0090] On the other hand, the prepared nucleic acid-immobilized
substrate was mounted on a base of an apparatus for hybridization,
and hybridization was carried out by a conventional method (in
accordance with the instructions in the manual of the product of
Roche Diagnostics).
[0091] After completion of the hybridization, the nucleic
acid-immobilized fibers and the nucleic acid-immobilized substrate
were washed, and an enzyme-labeled anti-DIG antibody solution was
added to allow antigen-antibody reaction to occur. After the
reaction, the nucleic acid-immobilized fibers and the nucleic
acid-immobilized substrates were washed to remove the unbound
antibody. A DIG-detecting reagent was added and the mixture was
equilibrated. After draining water, optical signals were detected.
As a result, signals were detected depending on the immobilization
of the nucleic acid.
[0092] By this, it was confirmed that the apparatus for
hybridization according to the present invention may be used surely
as an apparatus for hybridization in the conventional method not
applying an AC voltage without a constitutional or functional
problem.
Example 2
Pretreatment of Optical Fibers and Substrate for Immobilizing
Selective Binding Substance
[0093] Each glass optical fiber having a diameter of 200 .mu.m
(produced by Hoya Shot) was cut with a special cutter for optical
fibers, and both end faces of the fiber were mirror-finished.
[0094] The thus processed optical fibers and a slide glass (76
mm.times.26 mm.times.1 mm) (produced by Matsunami Glass Kogyo) were
cleaned with a mixed solution of pure water, ethanol and NaOH, and
then washed with pure water. The cleaned surface was immersed in a
mixed solution of pure water and poly-L-lysine (composition: 10%
poly-L-lysine) to introduce amino groups to the surface of the
slide glass.
[0095] Using two types of nucleic acid solutions (".lambda.Control
Template & Primer Set-A" produced by Takara Shuzo Co., Ltd.;
Product No. TX803 (.lambda.DNA fragment of about 1000 bp) and
"Human TFR(1 kb) Template & Primer Set" produced by Takara
Shuzo Co., Ltd.; Product No. TX806 (DNA fragment of human
transferrin receptor of about 1000 bp), each of the nucleic acids
was amplified by PCR method. The primers used in the PCR method
were those contained in each commercial product. The amplification
products were purified to obtain purified nucleic acid solutions.
The two types of nucleic acid solutions were spotted on the surface
of the slide glass, to which amino groups were introduced, and the
resultant was subjected to UV crosslinking (120 mJ) after drying in
the air to obtain a nucleic acid-immobilized substrate in which two
types of nucleic acids were immobilized on the sites for
immobilizing nucleic acids. To block the remaining amino groups on
the slide glass surface, which did not react with the nucleic
acids, the surface on which the nucleic acids were immobilized was
immersed in a mixed solution of boric acid, pure water, NaOH for
adjusting pH, succinic anhydride and 1-methyl-2-pyrrolidone
(prepared by dissolving 3 g of succinic anhydride in 187 ml of
1-methyl-2-pyrrolidone, and 17 ml of 1M Na-borate (pH8.0) was added
immediately before use) and shaken, followed by washing the
surface.
Treatment of RNA
[0096] An RNA solution (".lambda.polyA+RNA-A) produced by Takara
Shuzo Co., Ltd.; Product No. TX802) was provided. This RNA has a
nucleotide sequence complementary to that of one of the
above-described nucleic acids (TX803). The RNA solution was mixed
with reverse transcriptase (Super script II, produced by GIBCO BRL;
Product No. 18064-071), 2.5 mM dATP, 2.5 mM dCTP, 2.5 mM dGTP, 1.0
mM dTTP, Cy5-dUTP (produced by Amersham Pharmacia; Product No.
PA55022), and the resulting mixture was incubated at 42.degree. C.
for 1 hour, to obtain a cDNA solution to which Cy5 dye was
incorporated.
[0097] Similarly, an RNA solution ("Human TFR RNA (1 kb)" produced
by Takara Shuzo Co., Ltd.; Product No. TX805) was provided. The
step of reverse transcription was carried out in the same manner as
described above except that Cy3-dUTP (produced by Amersham
Pharmacia; Product No. PA53022) was used in place of Cy5-dUTP to
obtain a cDNA solution in which Cy3 dye was incorporated. This cDNA
in which Cy3 dye was incorporated has a nucleotide sequence
complementary to that of one of the above-described nucleic acids
(TX806).
[0098] The two types of cDNA solutions to which the above-described
dyes were incorporated were mixed, purified and dissolved in a
buffer (3.4.times.SSC, 0.1% SDS) to obtain a solution for
hybridization.
Hybridization
[0099] Two optical fibers each in which one type of nucleic acid
was immobilized on one end face thereof and the cDNA solution
(solution for hybridization) containing the above-described dyes
were placed in a vinyl bag, and the bag was tightly closed such
that the solution for hybridization is not evaporated. The
resultant was left to stand at 65.degree. C. for 16 hours. The
optical fibers were then taken out of the vinyl bag and washed.
Detection of Fluorescence
[0100] Detection of fluorescence from Cy5 using the optical fiber
was carried out as follows. As the excitation light of the
fluorescence, laser (wavelength 635 nm) was used. The condensed
laser beam was irradiated on the end face of the optical fiber
immersed in the DNA solution, and the light emitted from another
end face was condensed a with condenser lens. A dichroic mirror
(produced by Omega Optical; Product No. XF2035) was arranged
downstream thereof at an angle of 45.degree. to the optical axis,
thereby removing extra excitation light. Immediately downstream of
the dichroic mirror, a bandpass filter (produced by Omega Optical;
Product No. XF3076) was arranged to further remove extra excitation
light. Immediately downstream of the bandpass filter,
Photomultimeter (produced by Hamamatsu Photonics; Product No.
H5784-02) was arranged and fluorescence from Cy5 dye was
observed.
[0101] To measure the fluorescence from Cy5 using the nucleic
acid-immobilized substrate, an optical system was prepared as
follows. As the excitation light of the fluorescence, laser
(wavelength 635 nm) was used. A bandpass filter (produced by Omega
Optical; Product No. X1069) was arranged perpendicularly to the
optical axis to remove extra light except for the excitation light.
A dichroic mirror (produced by Omega Optical; Product No. XF2035)
was arranged at an angle of 45.degree. to the optical axis of the
laser beam, and the condensed beam was irradiated on the end
surface of the optical fiber, which end surface is opposite to the
end surface immersed in the DNA solution. Further, the fluorescence
returned from an end surface immersed in the DNA solution was
condensed at the side of the end surface on which the excitation
light was irradiated, and the fluorescence was made to pass through
the above-mentioned dichroic mirror (produced by Omega Optical;
Product No. XF2035) and then the bandpass filter (produced by Omega
Optical; Product No. X1069) to cut extra excitation light.
[0102] In both cases using the optical fiber and the nucleic
acid-immobilized substrate, the fluorescence from Cy3 was detected
in the same manner as described above except that the dichroic
mirror and the bandpass filters were replaced with those for Cy3
(produced by Omega Optical; Product Nos. XF1074, XF2017 and XF3083,
respectively) and that the wavelength of the irradiated laser was
532 nm.
[0103] By these methods, the fluorescences from the two types of
optical fibers and from the nucleic acid-immobilized sites were
detected for Cy5 and Cy3. From the optical fiber prepared by
immersing the fiber in the nucleic acid solution of TX803,
fluorescence from Cy5 alone was observed, and fluorescence from Cy3
was not observed. From the optical fiber prepared by immersing the
fiber in the nucleic acid solution of TX806 and from the nucleic
acid-immobilized site prepared by spotting of the nucleic acid
solution of TX806, fluorescence from Cy3 alone was observed, and
fluorescence from Cy5 was not detected.
Example 3
[0104] The same procedures as in Example 2 were repeated except
that the optical fibers prepared were bundled, the beam of the
excitation light was scanned, and that fluorescence from each
optical fiber was detected. As a result, the same results as in
Example 2 were obtained.
Example 4
[0105] The same procedures as in Example 2 were repeated except
that the end face of the optical fiber, which is irradiated with
the excitation laser was the end face not immersed in the DNA
solution. That is, the procedures were the same as in Example 2
except that the direction of the optical fibers when detecting the
fluorescence was opposite to that employed in Example 2. As a
result, the same results as in Example 2 were obtained.
Example 5
[0106] Treatments of optical fibers and nucleic acids were carried
out in the same manner as in Example 2.
[0107] To measure the fluorescence from Cy5, the optical system was
constructed as follows (see FIG. 5). As the excitation light of the
fluorescence, laser 26 (wavelength 635 nm) was used. A bandpass
filter 19 (produced by Omega Optical; Product No. X1069) was
arranged perpendicularly to the optical axis to remove extra light
except for the excitation light. A dichroic mirror 20 (produced by
Omega Optical; Product No. XF2035) was arranged at an angle of
45.degree. to the optical axis of the laser beam, and the condensed
beam was irradiated on the surface of the slide glass opposite to
the surface immersed in the DNA solution. Further, the fluorescence
returned from an end surface immersed in the DNA solution was
condensed at the side of the end surface on which the excitation
light was irradiated, and the fluorescence was made to pass through
the above-mentioned dichroic mirror 20 (produced by Omega Optical;
Product No. XF2035) and then the bandpass filter 21 (produced by
Omega Optical; Product No. X3076) to cut extra excitation light. In
FIG. 5, reference numeral 22 denotes the condenser lens, 23 denotes
the optical fiber, 24 denotes a sample (DNA) and 25 denotes a
photomultiplier.
[0108] The fluorescence from Cy3 was detected in the same manner as
described above except that the dichroic mirror and the bandpass
filters were replaced with those for Cy3 (produced by Omega
Optical; Product Nos. XF1074, XF2017 and XF3083, respectively) and
that the wavelength of the irradiated laser was 532 nm.
[0109] By these methods, the fluorescence from the two types of
optical fibers was measured for Cy5 and Cy3. From the optical fiber
prepared by immersing the fiber in the nucleic acid solution of
TX803, fluorescence from Cy5 alone was observed, and fluorescence
from Cy3 was not observed. From the optical fiber prepared by
immersing the fiber in the nucleic acid solution of TX806,
fluorescence from Cy3 alone was observed, and fluorescence from Cy5
was not detected.
Example 6
[0110] As the optical fibers, image conduits (Produced by Edmont
Optics Japan; Product No. CJ53843) constituted by bundling a number
of thin optical fibers were used. This image conduit was formed of
3012 quartz optical fibers each of which had a thickness of 25
.mu.m.
[0111] The treatments of the optical fibers and the nucleic acids
were carried out in the same manner as in Example 5. The detection
optical system was the same as in Example 5. As a result, from the
optical fiber prepared by immersing the fiber in the nucleic acid
solution of TX803, fluorescence from Cy5 alone was observed, and
fluorescence from Cy3 was not observed. From the optical fiber
prepared by immersing the fiber in the nucleic acid solution of
TX806, fluorescence from Cy3 alone was observed, and fluorescence
from Cy5 was not detected. Taking the fluorescence intensities from
Cy5 and Cy3 in Example 5 are taken as 1, the fluorescence
intensities in this example were 3, so that the S/N ratio was
increased. The reason for this is thought to be that the condensed
light does not diverge in the optical fiber, and the excitation
light can be efficiently transmitted to the sample, when an image
conduit is used.
Example 7
Treatment of Optical Fiber
[0112] The glass optical fibers (produced by Hoya Shot) having a
diameter of 200 .mu.m used in Example 1 were coated with Pt film by
sputtering to obtain electroconductive optical fibers. The glass
optical fibers were cut with a special cutter for optical fibers,
and both end faces of the fibers were mirror-finished. The end face
of each optical fiber was immersed in a mixed solution of pure
water and poly-L-lysine (composition: 10% poly-L-lysine) to
introduce amino groups on one end face of each optical fiber.
[0113] The treatment of the nucleic acids and so on thereafter were
the same as in Example 2, to obtain two optical fibers each in
which one type of nucleic acid was immobilized on one end face
thereof, and the solution for hybridization.
Hybridization
[0114] The two optical fibers each in which one type of nucleic
acid was immobilized on one end face thereof, and the cDNA solution
(solution for hybridization) to which the dyes Cy5 and Cy3 were
incorporated were placed in a microtube (1.5 ml). A hole was formed
in the cap of the microtube, and the optical fibers were passed
through the hole. The end face of each optical fiber, on which the
DNA was immobilized was immersed in the solution for hybridization
in the microtube. The hole in the cap of the microtube was sealed
with paper bond such that the solution is not evaporated. The
volume of the solution for hybridization was 50 .mu.l. The solution
was one in which the labeled DNA was dissolved in pure water. The
resultant was left to stand at 65.degree. C. for 10 minutes. During
this, a voltage of 100V was applied such that the optical fibers
were used as the anode and a copper foil attached to the bottom of
the microtube was used as the cathode. The optical fibers were
taken out of the microtube and washed. The light was measured in
the same manner as in Example 2. As a result, from the optical
fiber prepared by immersing the fiber in the nucleic acid solution
of TX803, fluorescence from Cy5 alone was observed, and
fluorescence from Cy3 was not observed. From the optical fiber
prepared by immersing the fiber in the nucleic acid solution of
TX806, fluorescence from Cy3 alone was observed, and fluorescence
from Cy5 was not detected. The fluorescence intensities were the
same as those in Example 2. On the other hand, when the treatment
conditions of the optical fibers were the same as in Example 2
(i.e., electric voltage is not applied), and the hybridization time
was 10 minutes, the fluorescence intensities were 1/3 of those in
Example 2. Thus, it was proved that by making the optical fiber
electroconductive, and by applying electric field, even a period of
only 10 minutes is sufficient as the hybridization time.
Example 8
Pretreatment of Substrate for Immobilizing Selective Binding
Substance
[0115] To form electrodes on the selective binding
substance-arraying substrate, a stainless mask having two openings
each of which sized 10 mm.times.5 mm and which were arranged such
that the side of the 10 mm of each opening faces each other in
parallel via an interval of 10 mm was mounted on a slide glass, and
gold electrodes corresponding to the openings of the mask were
formed on the slide glass by sputtering method.
[0116] Thus prepared slide glass (76 mm.times.26 mm.times.1 mm)
(produced by Matsunami Glass Kogyo) on which the gold electrodes
were arranged was cleaned with a mixed solution of pure water,
ethanol and NaOH, and then washed with pure water. The cleaned
surface was immersed in a mixed solution of pure water and
poly-L-lysine (composition: 10% poly-L-lysine) to introduce amino
groups to the surface of the slide glass.
[0117] Using two types of nucleic acid solutions (".lambda.Control
Template & Primer Set-A" produced by Takara Shuzo Co., Ltd.;
Product No. TX803 (.lambda.DNA fragment of about 1000 bp) and
"Human TFR(1 kb) Template & Primer Set" produced by Takara
Shuzo Co., Ltd.; Product No. TX806 (DNA fragment of human
transferrin receptor of about 1000 bp), each of the nucleic acids
was amplified by PCR method. The primers used in the PCR method
were those contained in each commercial product. The amplification
products were purified to obtain purified nucleic acid solutions.
The two types of nucleic acid solutions were spotted on the surface
of the slide glass between the gold electrodes, to which amino
groups were introduced, and the resultant was subjected to UV
crosslinking (120 mJ) after drying in the air to obtain a nucleic
acid-immobilized substrate in which two types of nucleic acids were
immobilized on the sites for immobilizing nucleic acids. To block
the remaining amino groups on the slide glass surface, which did
not react with the nucleic acids, the surface on which the nucleic
acids were immobilized was immersed in a mixed solution of boric
acid, pure water, NaOH for adjusting pH, succinic anhydride and
1methyl-2-pyrrolidone (prepared by dissolving 3 g of succinic
anhydride in 187 ml of 1-methyl-2-pyrrolidone, and 17 ml of 1M
Na-borate (pH8.0) was added immediately before use) and shaken,
followed by washing the surface.
Treatment of RNA
[0118] An RNA solution (".lambda.polyA+RNA-A) produced by Takara
Shuzo Co., Ltd.; Product No. TX802) was provided. The RNA solution
was processed in the same manner as in Example 2 to obtain a cDNA
solution in which Cy5 dye was incorporated, and a solution for
hybridization.
Hybridization
[0119] The substrate for immobilizing nucleic acid, on which
surface the two types of nucleic acids were immobilized was fixed
on a base of the hybridization apparatus, and the gold electrodes
arranged on both sides of the nucleic acid-immobilized sites were
connected to means for applying AC voltage. On the sites on which
the two types of the nucleic acids were immobilized, 2 .mu.l of the
above-described solution for hybridization was dropped, and a cover
plate bridging the electrodes formed on both sides of the nucleic
acid-immobilized sites was placed. The cover plate was closed
tightly so as not to allow evaporation of the solution for
hybridization. An AC voltage of 10V, 10 Hz was applied between the
electrodes and the solution was incubated at 65.degree. C. for 10
minutes. Thereafter, the cover plate and the electrodes were
removed and washed.
Detection of Fluorescence
[0120] Fluorescences from Cy5 and Cy3 were measured using the same
optical system as in Example 2.
[0121] By these methods, the fluorescences from the two types of
the nucleic acid-immobilized sites were measured for Cy5 and Cy3.
From the nucleic acid-immobilized site prepared by spotting the
nucleic acid solution of TX803, fluorescence from Cy5 alone was
observed, and fluorescence from Cy3 was not observed. From the
nucleic acid-immobilized site prepared by spotting the nucleic acid
solution of TX806, fluorescence from Cy3 alone was observed, and
fluorescence from Cy5 was not detected.
[0122] The fluorescence intensities obtained from the nucleic
acid-immobilized substrate subjected to the application of the AC
voltage were comparable to those obtained by the conventional
methods in which the hybridization is carried out for a long time,
or in which a DC voltage is applied, even at a lower voltage.
Example 9
[0123] To examine the effect of the case wherein the electrodes are
not formed on the substrate for immobilizing selective binding
substance, but provided to the apparatus for selective binding
reaction, a slide glass (76 mm.times.26 mm.times.1 mm) (produced by
Matsunami Glass Kogyo) was cleaned with a mixed solution of pure
water, ethanol and NaOH, and then washed with pure water. The
cleaned surface was immersed in a mixed solution of pure water and
poly-L-lysine (composition: 10% poly-L-lysine) to introduce amino
groups to the surface of the slide glass. The immobilization of the
nucleic acids and treatments of RNA were carried out in the same
manner as in Example 8.
[0124] To confirm the effect of application of the AC voltage using
the substrate for immobilizing nucleic acid, the following
experiment was carried out. A substrate for immobilizing nucleic
acid on which two types of nucleic acids were immobilized on the
surface thereof was fixed on a base of the apparatus for
hybridization, and electrodes connected to means for applying AC
voltage of the apparatus for hybridization were arranged on both
sides of the region in which the nucleic acids were immobilized. In
this example, gold thin plates having a thickness of 0.15 mm were
used as the electrodes. The distance between the electrodes was 1
cm. On the sites on which the two types of nucleic acids were
immobilized, 20 .mu.l of the above-described solution for
hybridization was dropped, and a cover plate bridging the
electrodes on both sides of the nucleic acid-immobilized sites was
placed. The cover plate was closed tightly so as not to allow
evaporation of the solution for hybridization. An AC voltage of
10V, 10 Hz was applied between the electrodes and the solution was
incubated at 65.degree. C. for 10 minutes. Thereafter, the cover
plate and the electrodes were removed and washed.
[0125] For comparison with a conventional method in which AC
voltage is not applied, as a sample to be compared with the sample
to which AC voltage was applied, a sample obtained after incubation
at 65.degree. C. for 16 hours without applying a voltage between
the electrodes was provided. After incubation at 65.degree. C. for
16 hours, the cover plate and the electrodes were removed, and
washed.
Detection of Fluorescence
[0126] By the same method as in Example 8, the fluorescences from
Cy5 and Cy3 were measured. From the nucleic acid-immobilized site
prepared by spotting the nucleic acid solution of TX803,
fluorescence from Cy5 alone was observed, and fluorescence from Cy3
was not observed. From the nucleic acid-immobilized site prepared
by spotting the nucleic acid solution of TX806, fluorescence from
Cy3 alone was observed, and fluorescence from Cy5 was not
detected.
[0127] Further, the fluorescence intensities obtained from the
nucleic acid-immobilized substrate with which the AC voltage was
applied were about the same as those obtained when the electrodes
were provided to the apparatus for hybridization.
* * * * *